Abstract Memory-like or precursor exhausted (Tpex) CD8+ T cells are a critical reservoir in chronic infections and cancer, yet the signals sustaining their cytokine production remain unclear. Here, we identify KLRF1 as part of a CD4–CD8 communication axis that supports cytokine production in late-differentiated human CD8+ T cells. KLRF1 is upregulated in late-differentiated CD8+ T cells, and neutralizing KLRF1 reduces TNF and IFN-γ production. Differentiated CD4+ T cells express the KLRF1 ligand AICL, and in co-culture only AICL+ - not AICL⁻ - CD4+ T cells enhance cytokine output in CD8+ T cells. Using spatial proteomics of lung adenocarcinoma and adjacent tissue, we found that CD4+ AICL+ and CD8+ KLRF1+ T cells are enriched and spatially interacting in non-tumor regions, whereas both populations are reduced within tumor tissue. Single-cell RNA-seq of tissue samples and scRNA/ATAC analyses of circulating immune cells further showed that CD8+KLRF1+ T cells display a Tpex-like transcriptional and chromatin-accessibility profile. Together, these data identify the AICL–KLRF1 axis as a CD4+–CD8+ communication pathway that supports cytokine competence in late-differentiated CD8+ T cells.

The clinical outcome of acute myeloid leukemia (AML) is determined by therapy resistance and relapse. AML-induced immunosuppression has emerged as one of many cofactors and seems to be caused by distinct mechanisms, e.g. immune checkpoint interactions, altered antigen presentation and dysregulation of humoral factors. With chemotherapy, these alterations are expected to be reversible, but the exact mechanisms remain unclear. Adressing this issue, we performed single-cell RNAseq and spatial assessment to identify therapy-induced perturbations of the bone marrow (BM) microenvironment in newly diagnosed AML patients prior to and after induction therapy. 43,000 single-cell RNA sequencing profiles were generated from 12 paired samples in 6 AML patients using 10x Genomics 5'-scRNA/CITE-seq (140 Total-seq C antibodies) at initial diagnosis (T1) and after induction chemotherapy with either “7+3” or CPX-351 (T2). Analyses were performed using R v4.1.1 and Seurat v4.3.0. Signaling pathway activity was calculated using PROGENy. Stemness was estimated using CytoTrace. Potential cell-cell interactions were scored using CellChat. Spatial correlation was performed by histopathology and immunohistochemistry in matched trephine biopsies using the following stains: H&E, Giemsa, PAS, Gomori silver, Prussian Blue, Glycophorin C, MPO, CD61, CD34, CD117, CD14, CD68, CD20, CD3, CD138, VISTA. All slides were microscopically assessed by a trained hematopathologist. Pre-treatmentBM (T1) was characterized by reduction of normal hematopoiesis (p=0.009), in particular classical dendritic cells type 1 (cDC1) (p=0.066) and monocytes (p=0.034). High levels of AML cells were associated with significantly higher levels of mature naïve B cells (p=0.002), higher levels of class-switched B cells (p=0.030) and enrichment of inflammatory monocyte-like cells with MDSC phenotype. Regarding AML cells, 8 different transcriptional subtypes were identified across patients: 4 clusters with highly immature properties (undifferentiated and LMPP-like AML cells) and 4 clusters with signs of maturation (aberrant erythroid-like, EMP-like, promyelocyte-like and monocyte-like AML cells). The immature AML clusters showed high similarities in their transcriptional and signaling patterns, high stemness, quiescence, strong expression of homing factors (CD44, ITGA4), DNA instability and signs of chemoresistance such as upregulation of beta-Catenin, Trail and TGFb pathways. Furthermore, an upregulation of NFkB, TNFa and JAK/STAT signaling was observed in these AML cell clusters, indicating the formation of a highly inflammatory and dysfunctional immune microenvironment. This environment was characterized by a decrease of cDC1, presence of IFIT-high monocyte-like MDSCs, clonal expansion of senescent T cells as well as functional disturbation of antigen-presenting cells, T and NK cells with high expression of TGFb. Interestingly, each AML cell cluster showed a distinct expression profile of immune checkpoint markers, with most prominent expression of VISTA in monocyte-like AML cells. Spatially, VISTA-positive monocyte-like AML cells surrounded other AML cell populations in the BM niche. In addition, monocyte-like AML cells showed high expression of hypoxia- and metabolism-associated signatures (e.g. OXPHOS, fatty acid/cholesterol homeostasis), which might have supported the dysfunctional microenvironment. These microenvironmental changes were largely reversible after chemotherapy (T2). Our study reveals AML-induced dysregulation of the BM immune microenvironment, which seems reversible upon AML treatment with standard chemotherapy. Our data suggest that immature AML cells sustain the leukemic stem cell pool, whereas more mature EMP- and promyelocyte-like AML cells contribute to the bulk of AML. Furthermore, monocyte-like leukemic cells may shape the microenvironment to safeguard AML, possibly by VISTA-induced modulation of the immune system. This might explain limited therapeutic efficacy with currently available checkpoint inhibitors in AML. In the long run, identification of specific immune signatures in AML may improve our understanding of AML biology and inform therapeutic algorithms with regard to immunotherapeutic strategies in AML.

Therapy-induced acquired resistance limits the clinical effectiveness of mutation-specific KRAS inhibitors in colorectal cancer (CRC). Here, we investigated whether broad-spectrum, active-state RAS inhibitors meet similar limitations. We found that KRAS-mutant CRC cell lines were sensitive to the RAS(ON) multiselective RAS inhibitor RMC-7977, given that treatment resulted in RAS-RAF-MEK-ERK pathway inhibition; halted proliferation; and, in some cases, induced apoptosis. RMC-7977 initially reduced the activity of a compartment-specific, dual-color reporter of ERK activity, with reporter reactivation emerging after long-term dose escalation. These drug-resistant cell populations exhibited distinct patterns of phospho-protein abundance, transcriptional activities, and genomic mutations, including a Y71H mutation in KRAS and an S257L mutation in RAF1. Transgenic expression of KRAS G13D, Y71H or RAF1 S257L in drug-sensitive CRC cells induced resistance to RMC-7977. CRC cells that were resistant to RMC-7977 and harboring RAF1 S257L exhibited synergistic sensitivity to concurrent inhibition of RAS and RAF. Our findings demonstrate the power of reporter-assisted screening together with single-cell analyses for dissecting the complex landscape of therapy resistance. The strategy offers opportunities to develop clinically relevant combinatorial treatments to counteract the emergence of resistant cancer cells.

Study question What is the effect of placental invasion on the maternal cells in the decidua during early pregnancy? Summary answer Placental invasion shifts the maternal myeloid cell compartment toward a tolerogenic phenotype and induces complete decidualization in local fibroblasts. What is known already The maternal-fetal interface’s formation in early pregnancy is crucial for maternal and fetal health. Trophoblasts, specialized placental cells, infiltrate a region of the maternal mucosa known as decidua basalis (decB) while the decidua parietalis (decP) remains unaffected by this invasion. This study aims to create an atlas of the maternal-fetal interface in early human pregnancy and investigate the local cellular effects of trophoblast invasion. Study design, size, duration To investigate how placental invasion affects maternal decidual cells, uterine mucosal tissues from first-trimester pregnancy terminations were separated into decidua basalis, which is directly involved in placental invasion, and decidua parietalis, which is not. Decidua parietalis served as a within-patient control for the localized effects of placental invasion. Participants/materials, setting, methods Tissue samples from seven first-trimester pregnancies (weeks 7–8) were categorized into decidua basalis (decB) and decidua parietalis (decP). Donor-matched decP tissues served as internal controls, helping to differentiate localized trophoblast effects from systemic changes of pregnancy. The samples were then subjected to Xenium spatial transcriptomics, and isolated cells underwent CITE-seq using a customized antibody panel. Main results and the role of chance Placental invasion induced a pronounced shift within the myeloid compartment, and comparison with existing tumor datasets indicated that macrophage phenotypes at the invasion site closely resembled those seen in cancer—implying a shared physiological and evolutionary origin. Notably, fibroblasts in the decidua parietalis did not exhibit a fully decidualized phenotype, challenging prevailing models of decidualization; in contrast, fibroblasts in the decidua basalis were fully decidualized, a finding validated by flow cytometry, qPCR, and immunofluorescence. Remarkably, the decidua parietalis—where placental invasion is absent—featured immune “hubs” similar to those previously characterized in cancers, comprising CXCL10+ macrophages, T cells, specialized NK cells, and dendritic cells. Upon placental invasion, however, these hubs appeared to dissolve, accompanied by diminished T-cell and dendritic cell populations and a shift in NK cells toward an immune-tolerant phenotype. Taken together, these findings suggest that the placenta employs mechanisms analogous to those seen in tumors to establish local fetal tolerance. They also suggest a novel model for the process of decidualization, underscoring the dynamic interactions between placental cells and maternal fibroblasts before and after implantation. Limitations, reasons for caution Despite rigorously validating these findings through multiple methods, the limited sample size necessitates cautious interpretation of the results. Wider implications of the findings These findings highlight previously unrecognized parallels between the immunological processes at the maternal–fetal interface and those in tumor microenvironments. They offer insights into how the placenta may co-opt pathways commonly associated with cancers to establish local immune tolerance—information that could inform therapeutic strategies for pregnancy-related disorders. Trial registration number No

BACKGROUND:Distinguishing donor- vs. recipient-derived myelodysplastic neoplasm (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is challenging and has direct therapeutical implications. METHODS:Here, we took a translational approach that we used in addition to conventional diagnostic techniques to resolve the origin of MDS in a 38-year-old patient with acquired aplastic anemia and evolving MDS after first allo-HSCT. Specifically, we used single-cell transcriptional profiling to differentiate between donor- and recipient-derived bone marrow cells and established a strategy that additionally allows identification of cells carrying the MDS-associated U2AF1S34Y variant. RESULTS:The patient exhibited mixed donor chimerism combined with severely reduced erythropoiesis and dysplastic morphology within the granulocytic and megakaryocytic lineage along with the MDS-associated U2AF1S34Y mutation in the bone marrow. Single-cell transcriptional profiling together with targeted enrichment of the U2AF1S34Y-specific locus further revealed that, while the immune compartment was mainly populated by donor-derived cells, myelopoiesis was predominantly driven by the recipient. Additionally, concordant with recipient-derived MDS, we found that U2AF1S34Y-mutated cells were exclusively recipient derived with X but not Y chromosome-specific gene expression. CONCLUSION:Our study highlights the clinical potential of integrating high-resolution single-cell techniques to resolve complex cases for personalized treatment decisions. FUNDING:The study was funded by intramural resources of the Charité - Universitätsmedizin Berlin and the Berlin Institute of Health.

Single-cell analyses can be confounded by assigning unrelated groups of cells to common developmental trajectories. For instance, cancer cells and admixed normal epithelial cells could adopt similar cell states thus complicating analyses of their developmental potential. Here, we develop and benchmark CCISM (for Cancer Cell Identification using Somatic Mutations) to exploit genomic single nucleotide variants for the disambiguation of cancer cells from genomically normal non-cancer cells in single-cell data. We find that our method and others based on gene expression or allelic imbalances identify overlapping sets of colorectal cancer versus normal colon epithelial cells, depending on molecular characteristics of individual cancers. Further, we define consensus cell identities of normal and cancer epithelial cells with higher transcriptome cluster homogeneity than those derived using existing tools. Using the consensus identities, we identify significant shifts of cell state distributions in genomically normal epithelial cells developing in the cancer microenvironment, with immature states increased at the expense of terminal differentiation throughout the colon, and a novel stem-like cell state arising in the left colon. Trajectory analyses show that the new cell state extends the pseudo-time range of normal colon stem-like cells in a cancer context. We identify cancer-associated fibroblasts as sources of WNT and BMP ligands potentially contributing to increased plasticity of stem cells in the cancer microenvironment. Our analyses advocate careful interpretation of cell heterogeneity and plasticity in the cancer context and the consideration of genomic information in addition to gene expression data when possible.

PURPOSE:Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. So far, single-cell studies have been constrained by the requirement for prospectively collected fresh or cryopreserved tissue. This limitation might be overcome by recent technical developments enabling single-cell analysis of FFPE tissue. METHODS:We benchmark single-cell profiles from patient-matched fresh, cryopreserved and archival FFPE cancer tissue. RESULTS:We find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathway, hallmark, and clinically useful signatures, although expression of single genes varied due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting. CONCLUSION:Our analysis highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and increases the applicability in translational research.

Einleitung Im ersten Trimester der menschlichen Schwangerschaft treffen im Rahmen der Plazentation Zellen fetalen Ursprungs auf mütterliche Immunzellen. Spezialisierte Plazentazellen, genannt Trophoblasten, lösen sich von der Plazenta und invadieren die mütterliche Schleimhaut (Dezidua). Diese Zellen sind in Relation zum mütterlichen Immunsystem als semiallogen zu betrachten, da sie nur den halben Chromosomensatz mit der Mutter teilen. In bereits publizierten Studien unseres Labors konnte gezeigt werden, dass Trophoblasten über komplexe Mechanismen in der Lage sind, das Immunsystem lokal zu supprimieren, um so eine Immunreaktion zu vermeiden [1]. Um ein vollständigeres Bild der Effekte der Plazentation auf das lokale Immunsystem zu erhalten, haben wir folgende Studie durchgeführt.

Seven species of house geckos occur across the scattered islands of the Indian Ocean. Two of these, Hemidactylus frenatus and H. parvimaculatus are both widespread and possess distribution profiles that suggest pre-European, or perhaps natural dispersal to some islands. Of these, only H. frenatus currently has sufficient molecular data to begin exploring dispersal patterns. This species is one of the most successful reptile colonists, as demonstrated by its global, pantropical distribution. While in some areas, such as Australia and continental South America, its dispersal patterns are both recent and well-known, early historical records of Hemidactylus in the Indian Ocean islands suggest earlier and/or potentially non-human-mediated dispersals. Here, we reviewed the historical literature and combined those reports with an assessment of mitochondrial DNA diversity of a global sampling of H. frenatus samples that included modern and museum specimens. Our results corroborate previous studies and demonstrate the relatively high diversity within this species’ native range in Southeast Asia. In addition, the phylogenetic analysis suggests both a potential cryptic species complex, as well as global geographic structuring of different H. frenatus mitochondrial lineages. This has important implications for many comparative studies of this complex. Frequent and ongoing dispersals and colonizations complicate the identification of potentially older migration patterns. Further assessments including additional samples and analyses of additional genetic markers are necessary to disentangle older from more recent dispersals within this intriguing species.

The distribution of the black rat (Rattus rattus) has been heavily influenced by its association with humans. The dispersal history of this non-native commensal rodent across Europe, however, remains poorly understood, and different introductions may have occurred during the Roman and medieval periods. Here, in order to reconstruct the population history of European black rats, we first generate a de novo genome assembly of the black rat. We then sequence 67 ancient and three modern black rat mitogenomes, and 36 ancient and three modern nuclear genomes from archaeological sites spanning the 1st-17th centuries CE in Europe and North Africa. Analyses of our newly reported sequences, together with published mitochondrial DNA sequences, confirm that black rats were introduced into the Mediterranean and Europe from Southwest Asia. Genomic analyses of the ancient rats reveal a population turnover in temperate Europe between the 6th and 10th centuries CE, coincident with an archaeologically attested decline in the black rat population. The near disappearance and re-emergence of black rats in Europe may have been the result of the breakdown of the Roman Empire, the First Plague Pandemic, and/or post-Roman climatic cooling.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

Lung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread while no consensus treatment exists for metastasized carcinoids. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown. To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGFβ and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data. Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the non-inflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to define transcriptional states of colorectal cancer cells and signals controlling their development by performing single-cell transcriptome analysis of tumors and matched non-cancerous tissues of twelve colorectal cancer patients. We defined patient-overarching colorectal cancer cell clusters characterized by differential activities of oncogenic signaling pathways such as mitogen-activated protein kinase and oncogenic traits such as replication stress. RNA metabolic labeling and assessment of RNA velocity in patient-derived organoids revealed developmental trajectories of colorectal cancer cells organized along a mitogen-activated protein kinase activity gradient. This was in contrast to normal colon organoid cells developing along graded Wnt activity. Experimental targeting of EGFR-BRAF-MEK in cancer organoids affected signaling and gene expression contingent on predictive KRAS/BRAF mutations and induced cell plasticity overriding default developmental trajectories. Our results highlight directional cancer cell development as a driver of non-genetic cancer cell heterogeneity and re-routing of trajectories as a response to targeted therapy.

Since the 19th century, the addax (Addax nasomaculatus) has lost approximately 99% of its former range. Along with its close relatives, the blue antelope (Hippotragus leucophaeus) and the scimitar-horned oryx (Oryx dammah), the addax may be the third large African mammal species to go extinct in the wild in recent times. Despite this, the evolutionary history of this critically endangered species remains virtually unknown. To gain insight into the population history of the addax, we used hybridization capture to generate ten complete mitochondrial genomes from historical samples and assembled a nuclear genome. We found that both mitochondrial and nuclear diversity are low compared to other African bovids. Analysis of mitochondrial genomes revealed a most recent common ancestor ~32 kya (95% CI 11–58 kya) and weak phylogeographic structure, indicating that the addax likely existed as a highly mobile, panmictic population across its Sahelo–Saharan range in the past. PSMC analysis revealed a continuous decline in effective population size since ~2 Ma, with short intermediate increases at ~500 and ~44 kya. Our results suggest that the addax went through a major bottleneck in the Late Pleistocene, remaining at low population size prior to the human disturbances of the last few centuries.

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.

Abstract In colorectal cancer, oncogenic mutations transform a hierarchically organized and homeostatic epithelium into invasive cancer tissue lacking visible organization. We sought to identify differences in cellular composition between normal colon and colorectal cancer, and to define signals controlling cancer cell development. We used single cell RNA and protein profiling to analyze tumors and matched normal tissues of twelve colorectal cancer patients. RNA metabolic labelling followed by single cell RNA sequencing in patient-derived normal colon and colorectal cancer organoids was employed to define colorectal cancer cell developmental trajectories. We find that colorectal cancer tissues exhibited consistent changes in cellular composition in the epithelial, immune and stromal compartments across patients compared to normal colon. Tumor epithelial cells displayed patient-specific gene expression often correlating with somatic copy number alterations, but mainly organized into patient-overarching clusters. These clusters were defined by cell type-specific transcriptional programs related to stem, transient-amplifying and immature goblet cells, and showed differential expression for signatures of oncogenic traits such as replication stress. Patient-derived colorectal cancer organoids exhibited developmental trajectories forming along a gradient of mitogen-activated protein kinase activity that were Wnt-independent. Likewise, colorectal cancer cell types of patient samples were organized by mitogen-activated protein kinase activity. Our single-cell analyses provide a subtyping system for colorectal cancer cells based on transcriptional readout of morphogenetic signals and oncogenic traits. We provide evidence that mitogen-activated protein kinase signaling, a key pathway for targeted therapy, is a main driver of colorectal cancer cell plasticity.

Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.